Generating fast-twitch myotubes in vitro with an optogenetic-based, quantitative contractility assay

We here describe an in vitro contractility assay to control fiber-type composition in muscle cultures.


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We would like to thank the reviewers for their careful evaluation of our study. The goal of this work is to demonstrate that fiber type composition can be altered with exercise of in vitro muscle cultures. These findings provide an additional strategy to better mimic muscle in vitro for biological investigation and disease modelling. The reviewers' comments strengthened the conclusions of our study. In this point-by-point answer, we also include a statement on the status of each comment based on work we have performed since receiving the reviews.

Point-by-point description of the revisions
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Reviewer #1 (Evidence, reproducibility and clarity (Required)): The manuscript by Henning et al describes a method to induce myofiber subtype specification in vitro based on optogenetics and particle image velocimetry. The work is well performed and the manuscript is clear. The findings might be useful to the muscle community, but there are some issues which should be addressed in order to improve the quality and impact of the manuscript.
My main concern is that the whole work is performed in murine cells. Although I appreciate that the authors have used primary myoblasts rather than cell lines, I also think that the key advantage of such in vitro platforms is the possibility to "humanise" the experiments as much as possible. In this context, the key findings of this work should be reproduced using human myoblasts. This will significantly enhance the relevance of the work. Point 1.1) We thank the reviewer for the suggestion. We performed several pilot experiments to "humanize" our findings. We infected hiPSC-derived myotubes (van der Wal et al., 2018) and human immortalized myotubes (Mamchaoui et al., 2011) with either AAV9-pACAGW-ChR2-Venus-AAV or AAV1-pACAGW-ChR2-Venus-AAV to maximize infection efficiency. Unfortunately, human immortalized myotubes did not express ChR2 with either virus serotype, not permitting optogenetic training on these cultures. For hiPSC-derived myotubes, the infection rate was improved but insufficient to evaluate the effect of long-term intermittent light stimulation across the cultures. Moreover, the contractile behavior of hiPSC-derived myotubes expressing ChR2 significantly differed from primary mouse myotubes. Cells underwent a single and slow contraction when compared to the cyclic contractions observed in mouse myotubes. This suggests that the maturation of the contractile apparatus of 2D hiPSC-derived myotubes is insufficient to perform consistent in vitro training studies. As such, we agree with the reviewer that reproducing our key findings with human cells would improve the relevance of this work. However, due to the experimental limitations described above, significant improvements in human myotube maturation in vitro are required to perform such experiments. Our work is a proof of principal that fiber type composition can be influenced in vitro through contraction stimulation. We expect these findings to be the translated to human cultures when the field has discovered the necessary protocols to push human myotube maturation. Status: Failed due to technical limitations Other issues: 1) From a methodological perspective, I think some clarifications are needed on the western blots shown in Fig 4K-L, as the pattern of Myh3 and Myh8 in both panels appear very similar. This could easily be ruled out by providing raw data/images. Please accept my apologies if this is simply caused by similar migration patterns in the gels (worth checking).

Point 1.2)
The very similar appearance of both patterns is due to the same molecular weight (220 kDA) of distinct myh isoforms. After an initial staining of western blot membranes, primary and secondary antibodies were stripped off and the membrane was subsequently re-probed using a primary and secondary antibody. We incubated stripped membranes with secondary antibodies only and observed no signal, confirming the stripping was efficient. We have updated the representative images of the Western Blot membranes in Fig. 4K,L and Fig. S6 and included the α-actinin loading controls on which the bands are normalized to account for sarcomerogenesis. As stated in the manuscript, raw data will be available upon demand.

Status: Completed
2) Figure 3K-L (BTX): better imaging should be performed to assess morphology of NMJ (eg. pretzel-shaped as in mature/adult NMJ?) Point 1.3) We agree with the point raised by the reviewer. We performed a quantitative morphological evaluation of BTX stainings (Acetylcholine receptors) imaged with a confocal Full Revision 3 microscope ( Fig. 4SA-E). Additionally, we computed the number of BTX positive myonuclei. We measured no significant difference in NMJ morphology (size, roundness and circularity) but observed a 1.5-fold increase in the number of AChR clusters per myonucleus in trained myotubes (Fig. 3K). In general, a morphological assessment of the NMJ is difficult in this in vitro system due to our inability to generate mature muscle end plates with pretzel shapes as seen in in vivo adult NMJs.
We have included the new data into our manuscript ( Fig. 3K; Fig. S4A-E). Figure 3 N-P: Why did the authors used a relatively complex techniques such as smFISH to answer a question more simply addressable with more conventional (and perhaps less operator dependent) techniques such quantitative PCR?

3)
Point 1.4) qPCR analysis of ChrnE and ChrnG displayed insignificant expression changes between trained and untrained cultures despite exhibiting some trends. We reasoned that bulk approaches such as qPCR could abate differences due to the heterogeneity of our primary myotube cultures (presence of non-muscle cell types and varying degrees of muscle cell maturation). We therefore opted to monitor AChR expression at the single cell level in mature muscle cells, similar to those selected to perform the contraction analysis.
To better reflect this process, we inserted the qPCR data of ChrnE and ChrnG followed by the smFISH data in the manuscript. The new data was included in the supplementary information ( Fig. S4 F We thank the reviewer for his comments as the "other issues" raised will significantly improve the manuscript. With regards to using human myotubes, we were unable to apply long-term in vitro exercise in human cultures due to technical limitations. Nevertheless, it is our opinion that the main contribution of this manuscript is to show that fiber switching can be achieved in vitro and that this could be routinely used in the next generation of human in vitro muscle systems.
Comparison with other methods: Similar methods have been published but not with this level of resolution.

Full Revision
4 Expertise: muscle disease and regeneration, in vitro and in vivo models.
Reviewer #2 (Evidence, reproducibility and clarity (Required)): The work presented shows that muscle stem cells isolated from 5-day-old mice can be transduced with a DNA coding for a Channelrhodopsin2-Venus which will allow the muscle cell to be excited by a light beam (475nm) and to induce the contraction of myotubes. The authors measure the speed of contraction, relaxation and fatigability of such cells as a function of a more or less long excitation time. In particular, they show that myotubes in culture, excited at a frequency of 5 Hz, 8 hours per day for 7 days are larger than unstimulated myotubes and are more resistant to fatigue. Surprisingly, they show that myotubes stimulated at the low frequency of 5Hz express the neonatal Myosin heavy chain more than the slow Myh whose expression is known in adult muscle to be specifically strong in muscle fibers stimulated at low frequency. As the authors do not apply a high stimulation frequency (100Hz) to their culture, it is difficult to conclude whether the stimulation frequency applied in the study induces a specific phenotypic specialization of the myofiber, or a more general role. In this respect, the size of the myotubes obtained after training seems to be increased, showing a hypertrophic effect on the cultured myotubes. This study does not allow us to conclude, beyond the expression of the Myh8 gene, on the "gain" of the fast-twitch specialization of the myofiber by repeated stimulation over several days. A complementary study would certainly provide elements to better understand the role of muscle fiber stimulation, apart from the trophic contribution provided in vivo by the motoneuron. If the study is well conducted, some points are nevertheless important to address before publication.
Reviewer #2 (Significance (Required)): -Figures 4F/G are difficult to understand: the Myh7 signal seems much higher in trained myonuclei (F), but the histogram shows the opposite (G).

Point 2.1)
We apologize for the confusion. The apparent higher Myh7 signal in trained cells in Fig. 4F is due to background noise in the image. When mRNA is expressed, the smFISH probes are visible as fluorescent puncta. For clarity, we updated the representative images for the smFISH probes and highlighted the smFISH puncta with arrows. We also adapted the y-axis of each graph to better represent the analysis of mRNA counts. Status: Completed - Figures

Point 2.3)
We evaluated used myh antibodies by staining neonatal (5 days old pups) and adult muscle sections (TA, EDL, and Soleus). We indeed observed some issues with antibody specificity as rightfully highlighted by the reviewer. The antibody sc-32732 (Santa cruz) stained all fiber types contrary to the manufacturer´s notice. We therefore refer to these stainings as "total-Myh" in the manuscript. Antibody PA5-72846 (Thermo Fisher Scientific) is specific to neonatal (Myh8) but also all fast myh isoforms (Myh2, 1, and 4), termed "fast-Myh" in the article. For consistency, the slow-Myh7 antibody (A4.951; DSHB), which is specific for slow fibers, was renamed "slow-Myh". Despite these changes, our conclusions remain similar as we observe an increase in fast-Myh isoforms. To identify which fast isoforms account for the upregulation in fast_Myh, we performed western blots for Myh1, Myh2 and Myh4 (Fig. S5) as well as qPCRs (Fig. 4M) in trained and untrained cultures. We mostly observe a decrease in slow-Myh and an increase in fast-Myh4 at both protein and mRNA levels, confirming that long term in vitro exercise promotes a slow to fast myosin transition.
In the manuscript we have summarized our main findings for the antibodies that were used (sc-32732, PA5-72846, A4.951) in figure (Fig. S5), adapted the names, and updated Fig. 4K,L. The conclusion of a fiber type switch due to in vitro exercise was confirmed with qPCR (Point 2.4) and do remain valid. We greatly thank the reviewer for his expertise in the use of these antibodies.

Status: Completed
While 10Hz

Point 2.4)
The reviewer raises an important technical limitation of observing Myh proteins to identify fiber types due to the cross-reactivity of antibodies. Despite our best efforts to select and validate the appropriate antibodies, we agree that investigating mRNA expression of individual Myh isoforms would strengthen the conclusion of our study. We therefore designed specific primers and performed qPCR for distinct Myh isoforms on untrained and trained cultures. Using qPCR, we confirmed a fast-fiber type switch, as we observed a downregulation in slow-Myh7 and fast-Myh2 and an upregulation in fast-Myh1 (non-significant) and fast-Myh4 gene expression. Showing a trend in sequential fiber-type switching [slow-Myh7 → fast-Myh2 → fast-Myh1/4]. We have integrated this data in Fig. 4M. With regards to the "neonatal" phenotype of these in vitro cultures, this does indeed seem to be the case as the cultures mostly express developmental Myh3 and Myh8 isoforms but start to express the adult myosins (see Point 2.6 for more detail).

Status: Completed
Should we also be cautious about bulk analysis since, as shown in Figure S1, not all myotubes express ChR2? Point 2.5) Although 10% of myotubes do not express ChR2, we believe that 90% of infected myotubes is sufficient for bulk analysis. We nevertheless combine in our study bulk analysis with single cell assays such as smFISH and immunofluorescence, which are in line with the bulk analyses.

Status: Completed
May the authors correlate the ex vivo neonatal phenotype observed with the neonatal muscles they used to prepare myogenic stem cells? Point 2.6) We understand from this that the reviewer would like us to check the expression of distinct Myh isoforms in our in vitro system and compare it to neonatal muscle. For our primary cultures, we isolate myoblasts from the soleus, gastrocnemius, plantaris and quadriceps. Our qPCR data shows that Myh3 and Myh8 are the most highly expressed isoforms but adult myosins isoforms are present (see graph below). According to Agbulut et al. JBC 2003, which compared differential expression of Myh isoforms at different times after birth and in different muscles, our cultures compare to day 7 pups, which is similar to the age from which we isolate primary myoblasts.

Status: Completed
We thank the reviewer for his suggestions, which we have addressed. Those ensuring antibody specificity were particularly relevant to improve the manuscript.

Summary: In this work, the authors propose an in vitro model describing a strategy to alter fiber type composition of myotubes with a long-term, intermittent mechanical training. The authors present a model of myotubes transfected with an adenovirus, which makes them photosensitive; in this way, fibers contraction can be induced upon stimulation with blue LEDs. Even though ChR2 expressing myotubes have previously been used by other groups (Asano T, Ishizua T, Yawo H. Optically controlled contraction of photosensitive skeletal muscle cells.
Biotechnol Bioeng. 2012 Jan;109(1):199-204), no one has ever used it in the way proposed by qPCR performed on untrained, 4-day primary cultures for Myh genes and normalized on HPRT housekeeping gene. Note that lower Ct values reflect higher gene expression.

the authors. For this reason, this work opens new perspectives on the possible use for clinical and therapeutic purposes for this in vitro muscle system.
Major comments: I believe that the authors have presented their results, conclusion and methods in a fair and clear way, so that the experiment could also be reproduced. However, I think there are some adjustments that could be done in order to improve and strengthen the quality of this work: -The authors have analysed the expression of different myosin heavy chain isoforms, both regarding the slow and fast twitch fibers. Though, I think it would be interesting to investigate also the expression of Myh4, which is mainly expressed in type IIB fast twitch fibers; Point 3.1) We agree with the reviewer's comment. We added the analysis for Myh 4 (western blots and qPCR) to our manuscript ( Fig. 4M and Fig. S6). We have observed an upregulation of fast-Myh4 for trained myotubes. All new findings were discussed within the main text of the manuscript.

Status: Completed
The authors have observed a switch in the fiber type upon prolonged intermittent stimulation with blue LEDs, which translates into a higher number of type II fibers. It is known that exercise helps rescuing the loss of type II fibers, which is typical of age-related physiological processes, such as sarcopenia (Brunner F, Schmid A, Sheikhzadeh A, Nordin M, Yoon J, Frankel V. Effects of aging on Type II muscle fibers: a systematic review of the literature. J Aging Phys Act. 2007 Jul;15(3):336-48). However, I believe that providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic) could be helpful in order to have a clearer view on the specific subset of fibers that are generated with the given experimental conditions; Point 3.2) We performed lactate measurements in cell lysate and supernatant to monitor a switch from oxidative to glycolytic metabolism. We observed a non-significant increase in the lactate concentration from day 4 (2,92±0.84 M) to day 7 (3,89±0,33 M) of differentiation in the supernatant. However, we did not measure a significant difference between untrained and trained cultures. Additionally, we assessed protein level expression of oxidative phosphorylation complexes in mitochondria using western blots. We did not observe an altered level of assembly due to exercise. We have added the lactate measurements and OxPhos protein expression data in the supplementary information of the manuscript (Fig. S7). We used specific inhibitors of the glycolytic pathway (2-DG, UK5099, Rotenone and AntimycinA) as a control to prevent trained cells from shifting towards a fast fiber type. All inhibitors were tested at different concentrations and over distinct time periods (hours to days). In general, treated myotubes either became apoptotic or lost their ability to contract, therefore we were not able to perform our contractility-based mechanical training. The preliminary data was not included in the manuscript.

9
We hypothesize that the training-induced increase in contraction speed is primarily due to the expression of fast-myh isoforms. No metabolic changes were observed after 7 days of differentiation. However, we observed a non-significant trend of decreased protein expression for mitochondrial enzymes. To trigger a full fiber-type switch towards glycolytic myotubes, cells may need to be stimulated for longer culture periods.

Status: Completed
Minor comments: The text and the figures are clear and well written, and help to explain better the experimental setup and procedures. Still, I would suggest some minor adjustments: -I would suggest providing more information on the pH used for the experiments, since it plays a pivotal role in regulating myosin ATPase activity and, thus, muscular contractility. This would improve the replicability of your experiment.

Point 3.3.)
We thank the reviewer for this comment. We measured the pH at day 4 and 7 in medium of trained and untrained cultures. In both cases, pH was between 7.5-8. We added the information regarding the pH to the method and materials section "Primary mouse myotubes in vitro culture" in the supplementary information.

Status: Completed
The caption of Figure 1 is missing a description of panel E, even if it has been addressed in the text.

Point 3.4.)
We apologize for this mistake. We added the missing description of Fig. 1E.

This model opens new perspectives on in vitro muscle systems for the study of pathologies. The authors have been able to assess that myofibers contraction is able to induce a shift towards type II fibers, reproducing in vitro what is also known in vivo. For this reason, I believe that this model could be useful for further clinical approaches. It is important, though, to keep in mind that muscular disorders are not all characterized by a loss of type II fibers; for instance, myotonic dystrophies type I and type 2 exhibit similar phenotypes, even if different types of muscle fibers are affected. For this reason, it would be interesting to investigate the versatility of this model in terms of giving rise to different fiber types.
Point 3.5.) We added a sentence in the introduction that highlights an example of muscle disorders in which slow muscle fibers are predominately affected. Concerning the versatility of the model, we added a paragraph to the discussion elaborating on how different stimulus frequency and durations could influence the specialization of fiber types.

Status: Completed
Overall, the reviewer's comments greatly improved the manuscript. We were disappointed not to observe metabolic changes accompanying Myh switching but hope to revisit this concept in cultures with longer life spans.  Fig. 4M and Fig. S6. Point 3.2: providing a deeper analysis of the metabolism of the type II fibers (i.e. oxidative or glycolytic)we have performed lactate measurements and investigated the expression of mitochondrial enzymes via western blot, the new data can be found in Fig. S7 and was discussed in the main manuscript. Point 3.3: provide information of the pHwe added the pH of the media of untrained and trained cells to the materials and methods section "Primary mouse myotubes in vitro culture" in the supplementary information. Figure 1 is missing a description of panel E -We have added the missing description to the manuscript (Fig. 1E). Point 3.5: muscular disorders are not all characterized by a loss of type II fiberswe have added an example of a muscle disorder, in which slow fibers are predominantly affected, to the introduction of the manuscript. investigate the versatility of this model in terms of giving rise to different fiber typeswe added a paragraph to the discussion elaborating on how different stimulus frequency can lead to different fiber types.

Point 3.4: caption of
We have updated the Materials and Methods section to include all newly performed assays. The manuscript, figures, legends and supplementary information were updated following the formatting guideline of Development. All new findings have been integrated and discussed in the text of the main manuscript and representative figures have been added.

Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable. Point 1.1: Reproducing our key findings with human cellswe ran pilot experiments on immortalized human cell lines and human iPSC-derived myotubes but were not able to mature these cells sufficiently nor infect them to allow long-term in vitro training. Increased maturation of myotubes derived from hiPSCs is an endeavor currently undertaken by many laboratories. Although we agree that reproducing our key findings in human cells would increase the relevance of this manuscript, we believe the technical limitations are too important to address this point. Thank you for submitting your revised manuscript entitled "Generating fast-twitch myotubes in vitro with an optogenetic-based, quantitative contractility assay". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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